1. Field of the Invention
The invention relates to a device comprising a pressure generating assembly and a chamber for generating hydrostatic pressure in the said chamber, more particularly, a portable and autoclavable device for generating hydrostatic pressure in a fluid filled chamber receiving samples such as cell cultures, biological tissues, and cell seeded biomaterial constructs for the purpose of treatment with hydrostatic pressure.
2. Description of the Prior Art
Musculoskeletal disorders such as age-related degenerative changes of cartilage, intervertebral disc, collagen and bone contribute to some of the most common causes of impairment and disability for middle-aged and older persons, such as back pain, osteoarthritis, and degenerative joint and bone diseases. With an increasing aging population, it is therefore important to have a means for regeneration of articular cartilage, intervertebral discs as well as collagen and bone remodeling. Musculoskeletal disorders are multifactorial phenomenon and both mechanical and biological factors have been implicated in cases of accelerated degeneration.
During normal daily activities, the cartilage cells, the chondrocytes, in the cartilage of a diarthrotic joint experience levels of hydrostatic pressures in the order of 7 to 10 MPa (Hall et al., 1996) and increased cartilage thickness occurs in joint regions exposed to high intermittent hydrostatic stress (Wong et al., 1990). The intervertebral disc is routinely subjected to compressive loads that alter with posture and muscle activity and can produce pressures greater than 2 MPa in human lumbar discs in vivo (Wilke et al., 1999).
It has been shown by many studies that mechanical stimulation such as hydrostatic pressure is a major factor in maintaining load bearing tissues by influencing different biological factors at cellular level. Different studies have confirmed that hydrostatic pressure influences cellular response such a synthesis rate and increased collagen secretion by cells in major load bearing tissues such as cartilage (Smith et al., 2000) and intervertebral disc (Kasra et al. 2001, 3003, 2006).
In mechanical stimulation of intervertebral disc cells, Kasra et al. (2003) showed that within physiological levels of hydrostatic pressure up to 5 MPa, the magnitude of hydrostatic pressure was the dominant factor, and the higher the pressure the higher was the rate of protein synthesis by cells. The regenerative advantage of intermittent hydrostatic pressure at physiological levels 5-10 MPa applied to cartilage cells in vitro is also shown in U.S. Pat. Application Publication No. 20030133915A1. These studies suggest that for cell and tissue mechanical stimulation purposes related to tissue regeneration and in vitro studies of cell cultures, using hydrostatic pressures at high physiological levels of up to 10 MPa would be beneficial.
In most of research labs and commercial systems, hydrostatic pressure has usually been generated in an incubator by pressurizing the incubator gas in a vessel using a compressor. In an ordinary laboratory environment by pressurizing a gas in an incubator, it can be difficult to generate a high hydrostatic pressure, more than 1 MPa, and loading frequency is usually low and less than 1 Hz. For generating high hydrostatic pressures at high frequencies, Kasra et al. (2001) introduced a system for in vitro studies of cell cultures using a fluid filled cylindrical chamber receiving a cell culture and a piston moving in the said chamber to generate pressure, subjecting the cell culture to dynamic hydrostatic pressure. This system was operated by a servo-hydraulic external actuator and could generate pressures up to 5 MPa and 20 Hz frequencies. This method has been used in other studies such as the study reported by Le Maitre et al. (2009) related to therapy of intervertebral disc degeneration. A similar method was used in U.S. Pat. Appl. Pub. No. 20030133915A1 using a hydraulic loading instrument in fluid communication with the pressurizing chamber which could generate a dynamic hydrostatic pressure of 10 MPa at 1 Hz frequency. These fluid filled chambers operated by hydraulic systems are expensive, have a large size, not portable, and having difficulty of keeping a sanitary environment.
The concept of using a fluid filled cylindrical chamber receiving a biological tissue and a piston moving in the said chamber as reported by Kasra et al. (2001, 2003, 2005) was also used for generating very high static hydrostatic pressures up to 200 MPa for cryopreservation of cells and tissues described in U.S. Pat. Appl. Pub. No. 20070087321A1.
Ease of operation is also a major factor which has not been a priority in designing of the aforementioned hydrostatic pressure devices. For example, in U.S. Pat. Appl. Pub. No. 20070087321A1, the pressurizing chamber has two openings, one receiving a pressurizing piston and the other a pressure gauge. The pressure chamber is equipped with a cap for retaining and moving the piston inside the chamber by attaching the cap to the chamber using multiple screws. For relieving pressure in the chamber either the pressure gauge or the piston has to be removed. Removing and reinstalling the pressure gauge is not practical for multiple uses, especially if the chamber is of plastic type material, compromising the pressure gauge sealing. It is also time consuming to adjust the piston for inserting and removing the piston in and out of the chamber by screwing and unscrewing multiple screws on the cap. The system described in U.S. Pat. Appl. Pub. No. 20070087321A1 does not have any venting system and cannot be placed in an incubator and therefore not suitable for treating cell cultures in an incubator.
In most of the hydrostatic pressure devices, including the device of the U.S. Pat. Appl. Pub. No. 20070087321A1, a piston is driven directly into a pressurizing chamber containing the sample to be treated. Having the piston and the chamber of the same diameter has the disadvantage of having a limitation on the size of the chamber if the device is to be hand operated. The required load for pushing the piston in the chamber increases with the square power of the inner chamber diameter and as the inner chamber diameter increases, it increasingly becomes more difficult to operate the device by hand without any tool to generate a high hydrostatic pressure at 10 MPa level.
Sealing method is also a very important factor in the design of hydrostatic pressure devices, and it can influence the range of motion of the piston in the chamber and holding time of the pressure in the chamber as well as maintaining sealing quality after autoclaving. But there is hardly any details on sealing methods used in the disclosure of the aforementioned hydrostatic pressure devices.
Considering the advancement of the field of tissue regeneration and increasing use of hydrostatic pressure for cell and tissue mechanical stimulation, there is a need for a light, portable and easy to operate hydrostatic pressure device which can generate pressures at high physiological levels up to at least 10 MPa. This device needs to be autoclavable, quick and easy to operate by hand without any tool, and have a venting system which allows the device to be placed in an incubator without having to remove the samples from the chamber after each treatment. The ease of use and affordability of such a device is expected to provide most of laboratories with the opportunity of having an important tool for performing research in an important area of tissue regeneration and studying catabolic and anabolic responses of different cells to hydrostatic mechanical stimulation.